Telomeric noncoding RNA promotes mouse embryonic stem cell self-renewal through inhibition of TCF3 activity

Immunofluorescence staining. The cells were fixed in 4% parafor-maldehyde for 20 min and incubated at 37°C in blocking buffer (PBS containing 5% BSA and 0.2% Triton X-100). Then, the cells wereincubated in the presence of primary antibodies at 4°C overnight and were washed three times in PBS. Finally, the cells were incubated with an Alexa Fluor 488 (1:1,000, catalog no. A-11034, Invitrogen) or Alexa Fluor 594 (1:1,000, catalog no. A-11032; Invitrogen) secondary antibody for 1 h at 37°C. Nuclei were stained with Hoechst 33342 (1:10,000, catalog no. H1399; Invitrogen). The primary antibodies were against Nanog (1:500, catalog no. 14295-1-AP; Proteintech), Oct4 (1:200, catalog no. SC-5279; Santa Cruz), and SSEA1 (1:100, catalog no. SC-21702; Santa Cruz).Western blotting. mESCs were lysed in ice-cold RIPA cell buffer (Beyotime Biotechnology, China) supplemented with protease inhibitors (Transgene Biotech). Total protein was separated by a 10% PAGE gel (Bio-Rad) and was electrotransferred onto a polyvinylidene difluoride membrane. The primary antibodies used for probing were TCF3 (1:200, catalog no. SC-8635; Santa Cruz), FLAG (1:1,000, catalog no. F1804; Sigma), and α-tubulin (1:1,000, catalog no. SC-8035; Santa Cruz).The following secondary antibodies were used:goat anti-mouse IgG(H +L), horseradish peroxidase (HRP) conjugate(catalog no. HS201-01; Transgene Biotech) and donkey anti-goat IgG(H+L), HRP conjugate (catalog no. SC-2020; Santa Cruz). The signals were detected using Pro-Light HRP Kit (PA112-02, Tiangen Biotech, China). The density of the protein bands was quantified by ImageJ (NIH, Bethesda, MD).Accession number. Data and details of transcriptome-resequencing are available in the Gene Expression Omnibus under accession number GSE103933.Statistical analysis. All data are reported as means±SD. Student’s t-test was used to determine the significance of differences in comparisons. Values of P≤0.05 were considered as statistically significant.